Zhang X, Bremer H
Molecular Program, University of Texas at Dallas, Richardson 75083-0688, USA.
J Mol Biol. 1996 May 31;259(1):27-40. doi: 10.1006/jmbi.1996.0299.
The DNA-bending protein Fis of Escherichia coli is required for efficient initiation of chromosome replication and for activation of the P1 promoters of stable RNA (rRNA and tRNA) genes. Using fis+ and fis- bacterial strains, we have determined ribosome synthesis and activity, rRNA gene dosage, and transcript initiation rates at the rrnB P1 and P2 promoters. Different growth media were used to achieve growth rates between 0.7 and 2.7 doublings/hour for the fis+ strain, and between 0.7 and 2.2 doublings/hour for the fis- strain. In minimal media, the Fis deficiency only reduced the DNA and rrn gene concentration (DNA, oriC copies and rrn genes per protein); in amino acid-supplemented media, it also reduced rRNA synthesis rates per protein. Under all conditions, the ribosome activity (protein synthesis rate/ribosome) remained unchanged by the absence of Fis. In the presence of Fis, the absolute activities of the isolated rrnB P1 and P2 promoters increased from 2 to 88 and 10 to 50 initiations/minute, respectively, with increasing growth-rate. In the absence of Fis, these activities increased from 3 to 70 and 10 to 80 initiations/minute, respectively. Relative to the isolated rrnB P2 promoter, the strength of the rrnB P1 promoter was found to increase with increasing growth rate tenfold (from 0.17 to 1.7) in the presence, but only fivefold (from 0.17 to 0.85) in the absence of Fis. An evaluation of the data leads us to propose estimates of kcat and relative KM values for the two rRNA promoters, and relative values for free RNA polymerase concentrations during growth in different media. The analysis suggests that the reduced strength of P1 promoters of stable RNA genes in the absence of Fis, together with the reduced rrn gene concentration, increases the concentration of free RNA polymerase. In addition, the lower rrn P1 promoter activity in the absence of Fis reduces the probability that the downstream P2 promoter is blocked ("occluded") by a transcription elongation complex originating at the P1 promoter. The increased polymerase concentration and reduced P2 promoter occlusion both help to compensate for the Fis-deficiency, but during growth in rich media when the demand for ribosomes is high, this compensation is insufficient for fis- bacteria to achieve the wild-type level of ribosome synthesis and growth.
大肠杆菌的DNA弯曲蛋白Fis是染色体复制高效起始以及稳定RNA（rRNA和tRNA）基因P1启动子激活所必需的。利用fis⁺和fis⁻细菌菌株，我们测定了核糖体的合成与活性、rRNA基因剂量以及rrnB P1和P2启动子处的转录起始速率。使用不同的生长培养基使fis⁺菌株的生长速率达到每小时0.7至2.7代，fis⁻菌株的生长速率达到每小时0.7至2.2代。在基本培养基中，Fis缺乏仅降低了DNA和rrn基因浓度（DNA、oriC拷贝数以及每个蛋白质中的rrn基因数）；在补充了氨基酸的培养基中，它还降低了每个蛋白质的rRNA合成速率。在所有条件下，Fis缺失并未改变核糖体活性（蛋白质合成速率/核糖体）。在有Fis存在时，随着生长速率增加，分离出的rrnB P1和P2启动子的绝对活性分别从每分钟2次起始增加到88次起始以及从每分钟10次起始增加到50次起始。在没有Fis时，这些活性分别从每分钟3次起始增加到70次起始以及从每分钟10次起始增加到80次起始。相对于分离出的rrnB P2启动子，发现rrnB P1启动子的强度在有Fis存在时随着生长速率增加10倍（从0.17增至1.7），而在没有Fis时仅增加5倍（从0.17增至0.85）。对数据的评估使我们能够估算两个rRNA启动子的kcat和相对KM值，以及不同培养基中生长期间游离RNA聚合酶浓度的相对值。分析表明，在没有Fis时稳定RNA基因P1启动子强度降低，以及rrn基因浓度降低，会增加游离RNA聚合酶的浓度。此外，在没有Fis时较低的rrn P1启动子活性降低了下游P2启动子被源自P1启动子的转录延伸复合物“封闭”的概率。聚合酶浓度增加和P2启动子封闭减少都有助于弥补Fis缺乏，但在富含核糖体需求高的培养基中生长时，这种补偿不足以使fis⁻细菌达到野生型核糖体合成和生长水平。
